WANT TO IMPROVE YOUR WESTERN BLOTTING EFFICIENCY? TAKE ADVANTAGE OF OUR GREAT WORKFLOW OFFERS!
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1 WANT TO IMPROVE YOUR WESTERN BLOTTING EFFICIENCY? TAKE ADVANTAGE OF OUR GREAT WORKFLOW ERS!
2 Western blotting workflow GREAT ERS! Although the details of Western blotting protocols may vary from application to application, with adaptations to suit specific characteristics and the level of information required, they all follow these common basic steps: SAMPLE ELECTROPHORESIS TRANSFER PROBING DETECTION Since the introduction of the first enhanced chemiluminescent (ECL ) detection reagent for Western blotting, ECL, in 1990, th portfolio of products offered by GE Healthcare has been improved and optimised across all Western blotting requirements from electrophoresis and transfer equipment to highly sensitive detection systems and software.by selecting the optimal equipment, gels, markers, blockers, secondary antibodies, detection reagents, imaging systems and software, the GE Healthcare Western blotting portfolio enables you to easily achieve excellent results. What are your own Western blot analysis objectives? Great value Reproducible quantitive analysis High sensitivity What is the difference between qualitative and quantitative Western blotting? Qualitative analysis = verify the presence or the absence of a specific of interest Quantitative analysis = define the amount of on a blot either in relative or absolute terms Select those products below as identified by this Star rating to meet these analysis objectives The importance of good sample preparation cannot be stressed too highly. By understanding the nature of your starting sample and having a clear picture of the information you wish to derive from your Western blotting experiments, you increase your chances of a successful analysis. SAMPLE ELECTROPHORESIS TRANSFER PROBING DETECTION Almost all samples need further preparation after collection. Meticulous sample preparation is critical to successful analysis, so there is an absolute requirement to take particular care over this part of the process to ensure the best possible analytical results. It is not usually necessary to treat samples prior to 1-D gel electrophoresis, but it is very important in 2-D gel electrophoresis applications. However, if you experience problems with separation, such as blurred bands, sample cleanup improves performance by removing potentially interfering compounds such as nucleic acids, polysaccharides, and salts. Protein extraction 10% Protein purification 10% Mammalian Protein Extraction Buffer is designed for the efficient and gentle extraction of biologically active, total soluble s from mammalian cultured cells. This buffer is based on organic buffering agents and can be used for cell suspensions as well as adherent cells. Disposable PD-10 Desalting Columns offer an inexpensive and convenient alternative to lengthy and troublesome dialysis procedures. In minutes, biological samples are desalted and molecular weight solutes are removed or exchanged with excellent recoveries of macromolecules. Code GE Cat No Fisher Cat No Description Pack Size Promo price, Mammalian Protein Extraction Buffer 500mL Code GE Cat No Fisher Cat No Description Pack Size Promo price, PD-10 Desalting 30 columns
3 Electrophoresis is a separation technique based on the mobility of charged molecules in an electric field. It is used mainly for the analysis and purification of large molecules such as s or nucleic acids. Polyacrylamide gels Codes GE Cat No Fisher Cat No Description Function Pack Size Electrophoresis equipment - the MiniVE system Promo price, * PlusOne Acrylamide Page Monomeric unit of the gel matrix 1L * * PlusOne Methylenebis Acrylamide PlusOne Ammonium Persulphate Cross-linking agent for the formation of polyacrylamide Supports efficient polymerisation at concentrations as low as 1 mm 100g * 25g PlusOne Temed Polymerisation catalyst 25mL PlusOne Tris Commonly used as the solvent when preparing gels. With pka of 8.3 at ambient temperature, it has good buffering capacity in a ph range from 7 to 9 500g PlusOne Glycine Source of trailing ions, with pka of g PlusOne Glycerol PlusOne Bromophenol Blue A stabilising medium for casting polyacrylamide gradients. It also stabilises polyacrylamide gels for preservation. Electrophoresis tracking dye used in IEF, PAGE and sequencing. 25% 1L g *Buy more than three of these products and get 25% off. For everything else, get 15% off for any order. Why is it important to not overload s when running Western blots? Proteins concentrated at high density tend to self-associate through weak interactions rather than interacting with the membrane surface. As a result, :antibody complexes, the basis of the detection principle in Western blotting, are easily lost from the membrane during washing steps. Target size range (Mr) to to to to to % Recommended acrylamide concentration (%) - Perform 1-D and 2-D electrophoresis and electrotransfer in a single compact unit - Cast, run, and blot mini-gels in four streamlined components: gel module, blot module, lower buffer chamber, safety lid - Run two mini-gels or four mini-blots at once, using cm gel cassettes for increased separation distances - Complete semi-wet blotting in 45 min with only 300ml buffer - Choose precast gels from a variety of manufacturers - Perform two second-dimension separations using 7cm IPG strips in 1.5 h EPS 301 is the power supply of choice for the minive System. With ratings of 300V and 400mA, it is capable of both running and blotting gels. For faster transfers when blotting four gels at a time, use the EPS 2A200 for its higher current capability. Codes GE Cat No Fisher Cat No Description Promo price, MiniVE Complete 747, EPS301 Power supply 369,30 markers 50% Rainbow Molecular Weight Markers have been enhanced to enable faster and simpler identification of s on SDS-polyacrylamide gels. These ready-to-load markers provide sharper, more intense bands on gels and blots and have improved band spacing for more accurate molecular weight determination. The bright, distinctive colours of the markers allow easier confirmation of transfer to blotting membranes and orientation. ECL Plex Fluorescent Rainbow Markers are optimised for use with the ECL Plex Western Blotting Detection System and to provide visible marker bands on gels and membranes, as well as fluorescent images using Cy3 and Cy5 channels. M r Full-Range RPN800E Color Blue Red Green Yellow Purple Blue Orange Green Blue Red High-Range RN756E M r 10 3 Color 225 Blue 76 Yellow Purple 38 Blue 31 Orange 24 Green 17 Blue 12 Red 52 Codes GE Cat No Fisher Cat No Description Range (Mr) Visualisation N of s Pack Size Promo price, * RPN756E ECL Rainbow markers- High range to Visible, multicolor 8 250µL 83.30* * RPN755E ECL Rainbow markers- Low range to Visible, multicolor 7 250µL 87.08* RPN850E ECL Plex Rainbow markers to Fluorescent & visible, multicolour µL RPN851E ECL Plex Rainbow markers to Fluorescent & visible, multicolour µL RPN ECL DualVue Western blotting markers to Chemiluminescent, visible 3/7 215µL *Buy more than five of these products and get 50% off. Please note that this discount is not available if this minimum quantity is not reached. 3
4 Blotting equipment 25% Wet transfer is recommended for large s, but it is a relatively slow technique, requiring large volumes of buffer. Wet transfer should be applied in preference to semi-dry transfer when it is important to obtain blots of the highest quality in terms of distinct, sharp bands and efficient transfer. Semi-dry transfer is faster than wet transfer and, in addition, consumes less buffer. Heating is less of a problem with semidry transfer for normal transfer times, as the electrode plates adsorb heat, but semidry systems should be avoided for extended transfer times as this might lead to overheating and gel drying due to buffer depletion. Codes GE Cat No Fisher Cat No Description Transfer For Gel Size, cm Built-in Power Promo price, MiniVE Blotting Module Wet 9 x 10 N TE22 Mini Tank Transfer Unit Wet 9 x 10 N TE TE70 Semi-Dry Transfer Unit Semi-dry 14 x 16 N TE TE77 Semi-Dry transfer unit Semi-dry 21 x 26 N TE70 PWR Semi-Dry Transfer Unit Semi-dry 14 x 16 Y TE77 PWR Semi-Dry Transfer Unit Semi-dry 21 x 26 Y EPS301 Power suply When should I use wet transfer instead of semi-dry transfer? Wet transfer = recommended for large s. Preferred to semi-dry transfer when you wish to obtain high quality blots, distinct, sharp bands and efficient transfer. Semi-dry transfer = less efficient than wet transfer, especially for large s. Still, faster than wet transfer and, in addition, consumes a er volume of buffer. Using Rolls? Request your special price. blotting membranes 50% Optimised for chemiluminescent and fluorescent detection Excellent binding capacity over a wide size range Physical and chemical composition remains the same Please see selection on page 5. When choosing your membrane type, consider your sensitivity requirements, the molecular weight of of interest, detection method and if the application requires stripping and reprobing. Polyvinylidene difluoride (PVDF) membranes have high binding capacity, and as a consequence higher background. Recommended when high sensitivity is needed and in stripping and reprobing applications. Use of PVDF membranes with low autofluorescence properties is critical in fluorescence applications. Nitrocellulose membranes have lower binding capacity and as a consequence lower sensitivity and lower background. For most applications: use membrane pore size of 0.45µm For target s (Mr to ): er pore size (0.2µm) is recommended Codes GE Cat No Fisher Cat No Description Old name Membrane material Pore size, µm Protran Whatman Protran BA79 Nitrocellulose x 100 N 10 sheets Protran Whatman Protran BA83 Nitrocellulose x 100 N 10 sheets Protran Whatman Protran BA85 Nitrocellulose x 100 N 10 sheets Protran Premium Hybond ECL 0.2μm Nitrocellulose x 100 N 10 sheets Protran Premium Hybond ECL 0.45μm Nitrocellulose x 100 N 10 sheets Protran Supported Whatman Optitran BAS83 Nitrocellulose x 100 N 10 sheets Protran Supported Whatman Optitran BAS85 Nitrocellulose x 100 N 10 sheets Hybond P NEW PVDF x 100 N 10 sheets Hybond P Whatman Westran CS Size, mm Sandwich format Pack Size Promo price, PVDF x 100 N 10 sheets Hybond LFP Hybond LFP PVDF x 100 N 10 sheets Protran Whatman Protran BA79 Nitrocellulose x 90 Y 10 WB sandwiches Protran Whatman Protran BA83 Nitrocellulose x 90 Y 10 WB sandwiches Protran Whatman Protran BA85 Nitrocellulose x 90 Y 10 WB sandwiches Protran Premium Hybond ECL 0.2μm Nitrocellulose x 90 Y 10 WB sandwiches Protran Premium Hybond ECL 0.45μm Nitrocellulose x 90 Y 10 WB sandwiches Protran Supported Whatman Optitran BAS83 Nitrocellulose x 90 Y 10 WB sandwiches Protran Supported Whatman Optitran BAS85 Nitrocellulose x 90 Y 10 WB sandwiches Hybond P NEW PVDF x 90 Y 10 WB sandwiches Hybond P Whatman Westran CS PVDF x 90 Y 10 WB sandwiches Hybond LFP Hybond LFP PVDF x 90 Y 10 WB sandwiches * MM Chr Blotting paper x pieces 43.01* 10 precut membranes preassembled with 2 x 3MM Chr Blotting papers. Designed for convenience and ease-of-use. *Buy more than three of these products and get 30% off! 4
5 Selection guide for Western blotting membranes What is your detection method? Chemiluminescence Stripping and reprobing Fluorescence High-abundance High-abundance High-abundance s of a wide range of molecular s of a wide range of molecular s of a wide range of molecular Protran NC 0.1 Protran NC 0.2 Protran NC 0.45 Protran Supported NC 0.2 Protran Supported NC 0.45 Protran Premium NC 0.2 Protran Premium NC 0.45 Low-abundance protien Low-abundance Low-abundance s of a wide range of molecular s of a wide range of molecular s of a wide range of molecular 0.2 PVDF 0.45 PVDF 0.2 PVDF 0.45 PVDF 0.2 PVDF How does the binding capacity of the membrane affect my Western blotting results? The binding capacity of a membrane depends primarily on the pore size. A membrane with many pores has a larger binding surface than one with larger pores, and thus generally higher binding capacity. Although conformation and buffer composition also affect binding capacity, the overall sensitivity of a Western blot might depends on the amount of immobilised on the membrane and presented to the primary antibody. 5
6 Once your samples are separated and transferred onto a membrane, the of interest is detected and localised using a specific antibody. blocking agent 15% Blocking is an important step to allow an efficient detection of the of interest. What is important to consider when selecting a blocking reagent? As nonspecific binding of antibodies to the membrane is detrimental to the specificity and sensitivity of the assay, it is essential to block spaces not already occupied by s. Your choice of blocking strategy will be guided by your samples and the antibodies used. No single blocking agent is ideal for every Western blotting process. Determining the optimal blocking agent and concentration are key steps for successful immunodetection. GE Cat No Fisher Cat No Description Pack Size Promo price, Codes RPN ECL blocking agent 40g RPN ECL Prime blocking reagent 40g ECL reagent HRP Detection reagent Detection reagent reacts reacts with with HRP HRP and and generates light light emission What can I do to prevent high background? High background, where nonspecific binding is masking the signal from the of interest, is most commonly an issue related to the blocking step or dilution of antibodies. Causes include: An inappropriate blocking reagent. Insufficient blocking or overly extensive washing after the blocking step. Too high concentration of the secondary antibody followed by insufficient washing. High background is best prevented by careful optimisation of blocking conditions and antibody concentrations. HRP-conjugated secondary antibody recognizes recognises the the primary antibody primary antibody Proteins on membrane Proteins on membrane after after transfer transfer from from gel gel Primary Primary antibody antibody binds binds specifically to the to the target target ECL IgG, HRP-linked whole antibody 30% Codes GE Cat No Fisher Cat No Host Target Species Pack Size Promo price, * NA9311ml Sheep Mouse 1 ml * NA931100ul Sheep Mouse 100 µl * NA9341ml Donkey Rabbit 1 ml * NA934100ul Donkey Rabbit 100 µl *Buy more than three of these products and get 30% off! What should I consider when selecting primary antibodies? Consider how the of interest is folded in your specific application, as different epitopes will be exposed under different conditions. Use antibodies developed for Western blotting purposes, e.g. antibodies generated by using a denatured antigen, if you run a traditional Western blot. If native conditions are used, select an antibody where a native antigen was used for immunisation. How can I optimise the dilution of my antibodies? The optimal antibody concentration is usually determined by testing a series of antibody dilutions spanning two-fold dilutions either side of the recommended dilution, e.g. : 1:25, 1:50, 1:100, 1:200, and 1:400 for a recommended 1:100. To further optimise the probing conditions, the same titration may be performed for extended or reduced times, such as 1, 2 and 3 hours at room temperature or overnight at 4 C. ECL Plex CyDye -Conjugated Antibodies ECL Plex Western Blotting System is optimised for single detection as well as multiplex detection using ECL Plex CyDye-Conjugated Secondary Antibodies and Hybond-LFP (low-fluorescent PVDF membrane) or Hybond ECL (nitrocellulose membrane). The optimised antibodies show high sensitivity, a very good dynamic range and low or no cross-reactivity. CyDye technology is dependable, reliable, and ensures accurate quantitation. Codes GE Cat No Fisher Cat No Host Target Species Conjugate Pack Size Promo price, Goat Rabbit Cy3 150µg PA Goat Mouse Cy3 150µg
7 Western blotting detection reagent 30% To celebrate the 25th anniversary of ECL, GE launched ECL start a reliable chemiluminescence WB substrate. Enhanced chemiluminescent (ECL) detection is based on antibodies conjugated to horseradish peroxidase (HRP). HRP catalyses the oxidation of luminol in the presence of peroxide, generating emission of low intensity light at 428nm. The signal intensity is dependent of the number of HRP molecules, and accordingly proportional to the amount of antibody bound to the target molecule. Codes GE Cat No Fisher Cat No Description Applications Pack Size Promo price, RPN ECL start For multiple exposures of the membrane, or for 2000cm² membrane large time window between experiment and RPN ECL start analysis due to a long signal duration for 4000cm² membrane RPN ECL RPN ECL Prime RPN ECL Select For reliable detection of high to medium abundant s For quantitative analysis of high to low abundant s For quantitative analysis of medium to very low abundant s for 4000cm² membrane for 1000cm² membrane for 1000cm² membrane Let s celebrate 25 years of ECL... Order your ECL start now! Use the guide below to help choose a detection reagent depending on your experimental design... Type of analysis Detection method Protein expression level Experimental characteristics Recommended detection reagent High ECL Start X-ray film Medium ECL Low/Very low ECL Select Verify presence of High ECL Start CCD imager Medium ECL Low/Very low Large experimental setup ECL Prime Scarce antibodies ECL Select High ECL Start Quantitative analysis CCD imager Medium Large experimental setup ECL ECL Prime Low/Very low Scarce antibodies ECL Select 7
8 Hyperfilm High sensitivity for low concentration of nucleic acid and targets Optimised for use in all blue and green light chemiluminescence systems Clear background for excellent contrast and improved band resolution Publication-quality images 35% Buy more than three of any of these products and get an extra discount of 35% instead of 10%! Codes GE Cat No Fisher Cat No Description Dimensions, cm Dimensions, inches Pack Size Promo price, Hyperfilm ECL sheets Hyperfilm ECL sheets Hyperfilm ECL sheets Hyperfilm ECL sheets Hyperfilm ECL sheets What is important to consider when detecting chemiluminescence signals with X-ray film? X-ray film has limitations for quantitative analysis. Compared to CCD imaging, X-ray film imaging has limited linear dynamic range, and accurate detection of both weak and strong signals on the same film is not possible. Furthermore, X-ray film processing carries additional requirements for both a darkroom and facilities for disposal of waste solutions, etc. After X-ray film development, a digital image must also be created if further data analysis is required or if the image is to be published. HERE ARE THE MOST POPULAR STAR PRODUCTS - CHOOSE THE BEST PACKAGE FOR YOUR OWN LAB! Great value ECL start Western blotting detection reagent ECL Rainbow marker - high range ECL blocking agent, 40g Protran 0.2 NC Quantitative analysis ECL plex Cy3 anti-rabbit ECL plex Cy3 anti-mouse ECL Plex Rainbow marker 120µl ECL Prime blocking agent Hybond LFP 0.2µm High sensitivity ECL Select Western blotting detection reagent ECL DualVue Western blotting marker ECL Prime blocking agent 0.2µm PVDF Want to try? Request a sample from your local representative. Prices valid until 30th June 2016 Offers only valid when Fisher promotion codes are used when ordering. Offers apply to some products only when minimum order requirements are met. Note that not all products included in the brochure are available as samples Thermo Fisher Scientific Inc. All rights reserved. Trademarks used are owned as indicated at Austria: +43(0) Belgium: +32 (0) Denmark: Germany: +49 (0) Ireland: +353 (0) Italy: Finland: +358 (0) France: +33 (0) Netherlands: +31 (0) Norway: Portugal: Spain: Sweden: Switzerland: +41 (0) UK: +44 (0)
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